Suzuki Atsushi, Sekiya Sayaka, Onishi Makiko, Oshima Naoko, Kiyonari Hiroshi, Nakauchi Hiromitsu, Taniguchi Hideki
Division of Organogenesis and Regeneration, Post-Genome Science Center, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
Hepatology. 2008 Dec;48(6):1964-78. doi: 10.1002/hep.22558.
The adult liver progenitor cells appear in response to several types of pathological liver injury, especially when hepatocyte replication is blocked. These cells are histologically identified as cells that express cholangiocyte markers and proliferate in the portal area of the hepatic lobule. Although these cells play an important role in liver regeneration, the precise characterization that determines these cells as self-renewing bipotent primitive hepatic cells remains to be shown. Here we attempted to isolate cells that express a cholangiocyte marker from the adult mouse liver and perform single cell-based analysis to examine precisely bilineage differentiation potential and self-renewing capability of these cells. Based on the results of microarray analysis and immunohistochemistry, we used an antibody against CD133 and isolate CD133(+) cells via flow cytometry. We then cultured and propagated isolated cells in a single cell culture condition and examined their potential for proliferation and differentiation in vitro and in vivo. Isolated cells that could form large colonies (LCs) in culture gave rise to both hepatocytes and cholangiocytes as descendants, while maintaining undifferentiated cells by self-renewing cell divisions. The clonogenic progeny of an LC-forming cell is capable of reconstituting hepatic tissues in vivo by differentiating into fully functional hepatocytes. Moreover, the deletion of p53 in isolated LC-forming cells resulted in the formation of tumors with some characteristics of hepatocellular carcinoma and cholangiocarcinoma upon subcutaneous injection into immunodeficient mutant mice. These data provide evidence for the stem cell-like capacity of isolated and clonally cultured CD133(+) LC-forming cells.
Our method for prospectively isolating hepatic progenitor cells from the adult mouse liver will facilitate study of their roles in liver regeneration and carcinogenesis.
成体肝祖细胞在几种类型的病理性肝损伤时出现，特别是当肝细胞复制受阻时。这些细胞在组织学上被鉴定为表达胆管细胞标志物并在肝小叶门区增殖的细胞。尽管这些细胞在肝再生中起重要作用，但将这些细胞确定为自我更新的双能原始肝细胞的确切特征仍有待揭示。在此，我们试图从成年小鼠肝脏中分离出表达胆管细胞标志物的细胞，并进行基于单细胞的分析，以精确检查这些细胞的双谱系分化潜能和自我更新能力。基于微阵列分析和免疫组织化学的结果，我们使用抗CD133抗体并通过流式细胞术分离CD133(+)细胞。然后，我们在单细胞培养条件下培养和扩增分离的细胞，并检查它们在体外和体内的增殖和分化潜能。在培养中能够形成大集落（LCs）的分离细胞产生肝细胞和胆管细胞作为后代，同时通过自我更新细胞分裂维持未分化细胞。形成LC的细胞的克隆后代能够通过分化为功能完全的肝细胞在体内重建肝组织。此外，在分离的形成LC的细胞中缺失p53导致在皮下注射到免疫缺陷突变小鼠中时形成具有肝细胞癌和胆管癌某些特征的肿瘤。这些数据为分离和克隆培养的CD133(+)形成LC的细胞的干细胞样能力提供了证据。
我们从成年小鼠肝脏中前瞻性分离肝祖细胞的方法将有助于研究它们在肝再生和致癌作用中的作用。
